Mass spectrometry (MS) is a powerful qualitative and quantitative analytical technique that has been introduced into many clinical and research laboratories during the last 5 years. The cost of MS analyzers has dropped to a range that is affordable for a majority of laboratories. In the clinical laboratory, mass spectrometers are used to measure a wide range of clinically relevant analytes. When applied to biological samples, the power of MS lies in its selectivity toward the identification and quantification of compounds. Tandem MS (MSMS) is a MS technique which offers excellent analytical capabilities.
Screening such as newborn screening for inherited metabolic disease, congenital hypothyroidism, haemoglobinopathies, cystic fibrosis, and a range of other conditions defined by local requirements is now mandatory in many countries.
Introduction of MSMS for metabolite screening has revolutionised both the analytical process and the number of conditions that, potentially, can be screened for. Typically this system involves butylation. However, screening using direct analysis of the metabolites is possible.
We have previously demonstrated the feasibility of detecting and confirming clinically significant haemoglobinopathies by MSMS in an analytical format suitable for newborn and ante-natal screening.
Metabolite screening allows detection of a whole range of metabolites that are diagnostic for particular inherited conditions. In some centres in the US and Europe up to 30 conditions have been included. However, for many conditions, the easily measured metabolites offer poor sensitivity and specificity, e.g. methionine for homocystinuria, which is a problem.
In the UK screening has been constrained to measuring phenylalanine for the diagnosis of phenylketonuria (PKU) and octanoylcarnitine for the diagnosis of medium chain acylCoA dehydrogenase deficiency (MCADD). This is a cost-effective approach. However, there is a desire to increase the screening profile, potentially excluding conditions that should be screened for, because of technical limitations, while including conditions with markers having low sensitivity and specificity. This prior art approach causes numerous logistical and clinical problems.
Biotinidase deficiency is an example of a condition that, at present, is not screened for using a metabolite. Biotinidase deficiency occurs in approximately 1 in 110,000 live births, depending on ethnic variations in the screened population. Undiagnosed biotinidase deficiency can result in death, anticonvulsant resistant fits, bilateral deafness, optic degeneration, gait problems, and a range of other non-specific clinical conditions. If diagnosed in the newborn period and treatment with pharmacological doses of biotin for life instituted the clinical phenotype is entirely suppressed and growth and development are normal. If diagnosed later in life some clinical manifestations may be controlled (e.g. fits), but others (e.g. deafness) remain. Consequently, despite its relatively low incidence in the population, it is a cost-effective screening candidate. In many centres outside the UK it is screened for using a separate test, measuring the enzyme activity directly, using a colorimetric assay based on measuring para-aminobenzoic acid released from biotinyl para-aminobenzoic acid (biotin PABA). The assay requires a dedicated separate sample (blood spot), is labour intensive, time consuming, and is relatively insensitive. No useful blood metabolite for diagnosis of biotinidase deficiency has been described. Thus, biotinidase screening according to the prior art involves numerous drawbacks and problems.
Type 1 tyrosinaemia is an example of a condition with a proposed marker having low sensitivity and specificity. Type 1 tyrosinaemia (fumarylacetoacetase deficiency) occurs in approximately 1 in 300,000 live births, depending on ethnic variations in the screened population. Undiagnosed type 1 tyrosinaemia can result in fulminant hepatic failure, coagulopathy, and death or rickets, porphyria like crises, neurological complications, and hepatic tumours. Hepatic transplant is often the only treatment. If diagnosed in the newborn period and NTBC treatment for life is instituted, the clinical phenotype is entirely suppressed and growth and development are normal. Consequently, despite its rarity, it is considered a cost-effective screening candidate. In many cases, blood tyrosine is increased, but this is not diagnostic and non-specific increases in blood tyrosine are common in the newborn period, making it problematic to attempt to use blood tyrosine level as a sole indicator of the disease. Succinylacetone is considered to be the diagnostic metabolite but its measurement does not integrate easily into the prior art metabolite screening systems, which is a problem. Succinylacetone was first recognised because of its inhibition of the enzyme porphobilinogen synthase (PBG synthase links two 5-aminolevulinic acid molecules by dehydration), inhibition of which contributes to the porphyria-like crises. In many centres porphobilinogen synthase activity is used to screen for type 1 tyrosinaemia or provide a back up test for initial indications of increased tyrosine. The assay is based on providing 5-aminolevulinic acid as substrate and measuring the porphobilinogen produced. The assay requires a separate blood spot, is labour intensive, time consuming, and is relatively insensitive. Lack of understanding of the enzymatic process also raises problems in the prior art connected with tyrosinaemia.
Enzyme activities are usually measured under standardised and optimised conditions. Such standardisation/optimisation requires specific buffers and pH conditions. Consequently, individual assays and conditions are almost always different, often strikingly so. Thus, each assay is almost unique. Assay conditions for a first enzyme of interest are rarely suitable for a second or further enzyme of interest, and indeed can even inhibit or inactivate it. These are serious problems when it is desired to perform multiple enzymatic analyses.
In addition, it is known that buffers can significantly suppress electrospray ionisation of important compounds. This is a problem in the art. Prior art attempts to solve this problem typically employ chromatography to remove the buffer from the compound signal. This is a time consuming and labour intensive step requiring specialist equipment and knowledge to carry out.
The present invention seeks to overcome problems associated with the prior art.